Most drugs, xenobiotics and environmental pollutants that enter the human body are metabolized by P450 enzymes in the endoplasmic reticulum of liver cells. Such microsomal type II P450 enzymes, which include several enzymes involved in adrenal and gonadal steroid hormone synthesis, require electron donation from P450 oxidoreductase (POR) to mediate catalysis. POR is a 680 amino-acid, 78 kDa protein that contains two flavins, a flavin adenine dinucleotide moiety (FAD), and a flavin mononucleotide (FMN). There is substantial genetic variation in the hepatic P450 enzymes. This variation, plus the genetic variation in drug transporters and receptors, accounts for some, but not all, genetic variation in drug metabolism. We propose that some of this unexplained genetic variation in drug metabolism may be explained by genetic variation in POR. Our laboratory discovered POR deficiency as a new disorder of steroid hormone biosynthesis in 2004. Through analysis of patient DNAs, and sequencing of a large cohort of normal individuals, we have identified 35 POR amino acid sequence variants. In our recent study of 842 normal individuals, we discovered that nearly 28% of human POR alleles carry the A503V sequence variant. In vitro assays show that A503V has decreased activities in assays based on cytochrome c and P450c17, but not in assays based on CYP1A2 and CYP2C19. Assays of the activities of these POR missense mutants with cytochrome c, steroidogenic P450c17 (CYP17), and drug-metabolizing CYP1A2 and CYP2C19 have shown that the activity of a particular POR mutant with one target P450 may not predict its activity with another P450 enzyme. Thus the analysis of the potential role of POR sequence variants in the pharmacogenetics of drug metabolism is both important and complicated. We hypothesize that genetic variants of POR contribute to variation in human drug metabolism and that mildly defective POR sequence variants may synergize with otherwise innocuous, mildly defective variants in hepatic P450 enzymes to cause important variations in drug metabolism. To begin to characterize the contributions of POR to human pharmacogenetics, we propose three specific aims. Aim 1 is to determine the catalytic activities of the A503V POR variant and the common disease-causing POR mutants A287P and R457H with hepatic, drug-metabolizing P450 enzymes. Aim 2 is to determine whether the A503V variant has selective loss (or gain) of activity with common variants of hepatic, drug-metabolizing P450 enzymes. Aim 3 is to characterize the activity of the POR gene promoter and determine whether it contains polymorphisms that influence the level of POR expression. These studies will establish the in vitro biochemistry of POR variants in association with the principal drug-metabolizing hepatic P450 enzymes, and will permit us to design studies to determine whether the A503V variant affects drug metabolism in clinical studies in vivo. Completion of these aims will provide critically needed information about the potential role of POR in human pharmacogenetics. PUBLIC HEALTH RELEVANCE: There is substantial variation in how people of different ethnic backgrounds metabolize drugs; understanding such variations is crucial for optimizing drug dosing in clinical settings. Much of the variation between groups is based on variations in genes encoding drug transporters and drug-metabolizing enzymes. This project explores a third possibility, a gene called POR, that is needed for the activity of drug-metabolizing enzymes.